Lysine acetylation is a reversible post-translational modification (PTM) that affects protein function including stability, localization, and interaction with other proteins and DNA. Acetylation levels are regulated by the relative activity of histone N-acetyltransferases (HATs) and histone deacetylases (HDACs). Acetylated lysine side chains were first identified in histones, where they are proposed to regulate gene expression altering the accessibility to the DNA in nucleosomes. However, over 9000 acetylation sites have been identified in the mammalian proteome and these proteins have been implicated in a variety of cellular processes ranging from catabolism to cell homeostasis. Aberrant acetylation has been implicated in the development of T-cell lymphoma and several cancers, including gastric, prostate, colon, and breast cancers. Defining the substrate pool of each HDAC isozyme is critical for targeted drug development and understanding which pathways are affected by inhibition of each enzyme. HDAC8, the focus of this proposal, has been characterized and studied extensively in vitro, making HDAC8 an ideal model system for developing tools to identify HDAC interaction partners. HDAC8 activity toward acetylated peptides mimicking potential substrates has been measured; however, there are very few validated in vivo substrates. Recent mass spectrometry analyses have yielded a putative in vivo HDAC-protein interaction map, but it is not clear whether these interacting proteins are substrates or binding partners. The research in this proposal will bridge the gap between in vitro HDAC8 characterization and in vivo HDAC8 function and provide insight into in vivo HDAC8 substrate and binding partner interactions. I propose to (1) incorporate photo-active non-natural amino acids at different positions of HDAC8; (2) trap and analyze short and long-lived interacting partners and substrates with HDAC8 through mass spectrometry (in collaboration with Prof. B. Martin) and (3) validate substrates using an in vitro enzyme coupled assay. These experiments will facilitate identification of HDAC8 substrates. Development and application of these methods to other HDACs will lead to a better understanding of the HDAC interactome and development of HDAC-protein specific inhibitors.